A3 adenosine receptor ligand for use in treating ectopic fat accumulation

ABSTRACT

Provided is an A3AR ligand for reducing ectopic fat accumulation, particularly in fatty liver. Further provided is the use of A3AR ligand for the preparation of a pharmaceutical composition for reducing such fat accumulation, method of treating a condition associates with fat accumulation making use of the ligand and kits including pharmaceutical compositions including the ligand, and instructions for use of same, for treating a condition associated with ectopic fat accumulation. Further provided is the use of an A3AR agonist, such as 2-Chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (CI-IB-MECA, CF102) for treating fatty liver, specifically, non-alcoholic fatty liver disease (NAFLD).

TECHNOLOGICAL FIELD

The present disclosure concerns medical uses of A₃AR ligands.

BACKGROUND ART

References considered to be relevant as background to the presentlydisclosed subject matter are listed below:

International Patent Application Publication No. WO09/050707

International Patent Application Publication No. WO2013/111132

Acknowledgment of the above references herein is not to be inferred asmeaning that these are in any way relevant to the patentability of thepresently disclosed subject matter.

BACKGROUND

The Gi protein associated cell surface A₃ adenosine receptor (A₃AR), isover-expressed in cancer cells as well as in inflammatory cells and inperipheral blood mononuclear cells (PBMCs) derived from patients withvarious auto-immune inflammatory diseases, such as rheumatoid arthritispsoriasis and Crohn's Disease.

Activation of the Gi protein associated cell surface A₃ adenosinereceptor (A₃AR) with highly specific ligands, such as the A₃AR agonist2-Chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA)was found to induce hepatocyte proliferation as disclosed inInternational Patent Application Publication No. WO09/050707.

In addition, International Patent Application Publication No.WO2013/111132 describes the use of Cl-IB-MECA for treatment ofhepatocellular carcinoma (HCC) and for maintaining liver function in asubject having a chronic liver disease.

GENERAL DESCRIPTION

The present disclosure provides, in accordance with a first of itsaspects an A₃ adenosine receptor (A₃AR) ligand for use in reducingectopic lipid accumulation in a tissue of a subject.

The present disclosure provides, in accordance with a second aspect, amethod for reducing ectopic lipid accumulation in a tissue of a subject,the method comprising administering to said subject an amount of A₃ARligand.

The present disclosure provides, in accordance with a third aspect, theuse of an A₃AR ligand for the preparation of a pharmaceuticalcomposition for reducing ectopic lipid accumulation.

Further, the present disclosure provides, in accordance with a fourthaspect, a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and as active ingredient an A₃AR ligand in an amounteffective to reduce ectopic lipid accumulation in a tissue of a subject.

Finally, the present disclosure provides, in accordance with its fifthaspect, a kit comprising a pharmaceutical composition comprising an A₃ARligand and instructions for use of the pharmaceutical composition for inreducing ectopic lipid accumulation in a tissue of a subject.

In some embodiments, the A₃AR ligand is used for reducing fataccumulation in the liver.

In some further embodiments, the A₃AR ligand is an the A₃AR agonist,preferably 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide(Cl-IB-MECA, herein also referred to as CF102) for use in treating fataccumulation in the liver or a condition associated with fataccumulation in the liver of a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosedherein and to exemplify how it may be carried out in practice,embodiments will now be described, by way of non-limiting example only,with reference to the accompanying drawings, in which:

FIG. 1 is a bar graph showing the reduction of liver-to-body weightratio in NASH livers in mice following daily administration of CF102 forthree weeks as compared to non-treated mice (vehicle).

FIG. 2A-2B are bar graphs showing the decrease in plasma ALT level (FIG.2A) and in triglyceride level (FIG. 2B) in NASH mice following dailyadministration of CF102 for three weeks as compared to non-treated mice(vehicle).

FIG. 3A-3D are histological liver sections from the CF102 treated groupsat two different magnifications, ×50 and ×200 (FIGS. 3A and 3Brespectively) and vehicle (DMSO) treated group (FIGS. 3C and 3Drespectively).

FIG. 4 is a graph showing the decrease in NAFLD activity score followingtreatment with CF102 at a concentration of 200 μg/kg.

FIG. 5 is a graph showing the effect of CF102, at a concentration of 200μg/kg, in reducing the inflammation NAS score compared to the vehicle.

DETAILED DESCRIPTION

The present disclosure is based on the finding that2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA,herein also referred to as CF102), an A₃ adenosine receptor (A₃AR)agonist, with high affinity and selectivity to the A₃AR, inducedimprovement of non-alcoholic fatty liver disease (NAFLD) in a murineexperimental model. This unexpected improvement was exhibited, interalia, by the reduction of liver to body weight ratio in nonalcoholicsteatohepatitis (NASH) livers, by the decrease in triglycerides level inthe liver and most importantly, by the NAFLD score considered theendpoint (FIG. 4) and by the reduction in NAS inflammation score ascompared to the control vehicle (FIG. 5).

Based on these findings, it has been concluded by the inventors that anactivator of the A₃AR, be it an A₃AR agonist or an A₃AR allostericenhancer, is an effective tool for reducing fat/lipid accumulating inectopic sites, particularity and preferably in the liver.

Thus, in accordance with a first of its aspects, the present disclosureprovides an A₃AR ligand for use in reducing ectopic fat accumulation ina tissue of a subject having a condition associated with such fataccumulation, e.g. non-alcoholic fatty liver disease (NAFLD) orspecifically nonalcoholic steatohepatitis (NASH).

In the following description when referring to the A₃AR ligand for usein reducing ectopic lipid accumulation, it is to be understood as alsoencompassing a method for reducing ectopic lipid accumulation by theadministration of the A₃AR ligand; to the use of the A₃AR ligand for thepreparation of a pharmaceutical composition for reducing ectopic lipidaccumulation; to a pharmaceutical composition comprising apharmaceutically acceptable carrier and as active ingredient an A₃ARligand in an amount effective to reduce e lipid accumulation; and to akit comprising the pharmaceutical composition, and instructions for useof the composition for reducing ectopic lipid accumulation.

In the context of the present disclosure, when referring to lipidaccumulation it is to be understood as equivalently referring to fataccumulation. In the context of the present invention ectopic fataccumulation is used to denote deposition or storage of lipids,especially triglycerides in tissues and/organs other than adiposetissue, i.e. that under normal (healthy) conditions lack lipid cells(lipocytes/fat cells). Ectopic fat deposition is recognized as fataccumulation in the abdominal regions (as opposed tosubcutaneous/peripheral fat deposition) and is known to occur in theliver, skeletal muscle, heart and pancreas. Ectopic fat deposition isundesired and can cause health complications, such as insulinresistance, and thus its prevention or reduction is desired.

In a preferred embodiment, the A₃AR ligand is used for reducing lipidaccumulation in the liver.

In some embodiments, the reduction of lipid accumulation is in apopulation of subjects being diagnosed as suffering from NAFLD and/orNASH.

It is noted that NAFLD does not necessarily involve inflammation and itmay be that a fatty liver remains free of inflammation. It is furthernoted that in fatty liver, the liver functions normally and looks normalunder the microscope, except for accumulations of fat within cells. Inaddition, in NAFLD liver blood tests are typically either normal, orthere may be some slight increases in two of the enzymes made by theliver, the serum ALT (alanine aminotransferase) and/or the serum AST(aspartate aminotransferase).

The fat typically accumulating under condition of ectopic fataccumulation includes, without being limited thereto, glycerides(monoglycerides, diglycerides, triglycerides) and at times also sterolssuch as cholesterol.

In one embodiment, the reduction of lipid deposits is exhibited by atleast reduction in level of triglycerides (TG) in the tissue. In thiscontext, reduction in level of TG is to be determined as significantunder medical parameters. This may include, at times, at least 5%reduction in the level of TG as compared to the level thereof in atleast one earlier measurement time point (e.g. one or more days, weeksor months).

When the lipid deposited tissue is the liver, the reduction of lipiddeposition can also be exhibited by one of the following:

reduction in liver-to-body weight ratio;

reduced alanine aminotransferase (ALT) levels;

reduction in NAS score of inflammation.

In one embodiment, the A₃AR ligand is used for treating non-alcoholicfatty liver disease (NAFLD).

NAFLD is a condition in which fat accumulates in the liver of a patientwithout a history of alcohol abuse. NAFLD is classified into simplesteatosis and nonalcoholic steatohepatitis (NASH). In NASH, not onlysteatosis but also intralobular inflammation and hepatocellularballooning are present, often accompanied by progressive fibrosis. Inthe context of the present disclosure, any one of the above aretreating, i.e. simple steatosis, nonalcoholic steatohepatitis (NASH),intralobular inflammation, hepatocellular ballooning, progressivefibrosis, each constituting an independent embodiment in accordance withthe present disclosure.

Long-standing NASH may progress to liver cirrhosis, and hepatocellularcarcinoma (HCC) may be an outcome. Therefore, in some embodiments, bythe treatment of NAFLD, or specifically, NASH, the present disclosureprovides prevention of liver diseases, such as, liver cirrhosis, andhepatocellular carcinoma (HCC).

Accordingly, in one embodiment, the present disclosure provides the useof the A₃AR ligand for preventing liver disease in a subject being inpredisposition of developing said liver disease. The predisposition ofthe subject to develop a liver disease is determined by the presence offat accumulated in the liver or the existence of NAFLD.

In the context of the present disclosure “A₃ adenosine receptor ligand”or “A₃AR ligand” denotes any compound capable of directly (e.g. via thereceptor binding site) or indirectly (e.g. via an allosteric bindingsite) enhance the activity of the A₃ adenosine receptor, this includingfull or partial activation of the A₃ adenosine receptor. The A₃AR ligandis thus a molecule that exerts its prime effect through the enhancementof the activity of the A₃AR irrespective of whether the activation isvia the binding site or allosteric binding site. This means that at thedoses it is being administered it essentially affects only the A₃AR.

In one embodiment, the “A₃ adenosine receptor ligand” is an A₃ARagonist.

In one other embodiment, the “A₃ adenosine receptor ligand” is an A₃ARallosteric enhancer.

When referring to “A₃ adenosine receptor agonist” or “A₃AR agonist” itis to be understood to mean any ligand capable of specifically bindingto the A₃ adenosine receptor, thereby fully or partially activating theA₃ adenosine receptor. The A₃AR agonist is thus a molecule that exertsits prime effect through the binding and activation of the A₃AR. Thismeans that at the doses it is being administered it essentially binds toand activates only the A₃AR.

In one embodiment, an A₃AR agonist has a binding affinity (K_(i)) to thehuman A₃AR in the range of less than 100 nM, typically less than 50 nM,preferably less than 20 nM, more preferably less than 10 nM and ideallyless than 5 nM. Particularly preferred are A₃AR agonists that have aK_(i) to the human A₃R of less than 2 nM and desirably less than 1 nM.

However, it should be understood that some A₃AR agonists can alsointeract with and activate other receptors, however, with loweraffinities (namely a higher Ki).

A molecule will be considered an A₃AR agonist in the context of thepresent disclosure (namely a molecule that exerts its prime effectthrough the binding and activation A₃AR) if its affinity to the A₃AR isat least 3 times (i.e. its Ki to the A₃AR is at least 3 times lower),preferably 10 times, desirably 20 times and most preferably at least 50times larger than the affinity to any other of the adenosine receptors(i.e. A₁, A_(2a) and A_(2b)).

The affinity of an A₃AR agonist to the human A₃AR as well as itsrelative affinity to the other human adenosine receptors can bedetermined by a number of assays, such as a binding assay. Examples ofbinding assays include providing membranes containing a receptor andmeasuring the ability of the A₃AR agonist to displace a boundradioactive agonist; utilizing cells that display the respective humanadenosine receptor and measuring, in a functional assay, the ability ofthe A₃AR agonist to activate or deactivate, as the case may be,downstream signaling events such as the effect on adenylate cyclasemeasured through increase or decrease of the cAMP level; etc. If theadministered level of an A₃AR agonist is increased such that its bloodlevel reaches a level approaching that of the Ki of the A₁, A_(2a) andA_(2b) adenosine receptors, activation of these receptors may occurfollowing such administration, in addition to activation of the A₃AR. AnA₃AR agonist is thus preferably administered at a dose such that theblood level is such so that essentially only the A₃AR will be activated.

In one embodiment, the A₃AR agonist is a molecule that has a purinebackbone. In some embodiment, the purine containing compound may bedetermined as an A₃AR agonist based on acceptable structure-functionactivity assays.

The characteristic of some A₃AR agonists and methods of theirpreparation are described in detail in, inter alia, U.S. Pat. Nos.5,688,774; 5,773,423, 5,573,772, 5,443,836, 6,048,865, WO 95/02604, WO99/20284, WO 99/06053, WO 97/27173 and WO 01/19360, all of which areincorporated herein by reference.

According to some embodiments of the present disclosure, the A₃ARagonist is a purine derivative falling within the scope of the generalformula (I):

wherein,

R₁₁ represents an alkyl, hydroxyalkyl, carboxyalkyl or cyanoalkyl or agroup of the following general formula (II):

in which:

-   -   Y represents oxygen, sulfur or CH₂;    -   X₁₁ represents H, alkyl, R^(e)R^(f)NC(=O)—or HOR^(g)—, wherein        -   R^(e) and R^(f) may be the same or different and are            selected from the group consisting of hydrogen, alkyl,            amino, haloalkyl, aminoalkyl, BOC-aminoalkyl, and cycloalkyl            or are joined together to form a heterocyclic ring            containing two to five carbon atoms; and        -   R^(g) is selected from the group consisting of alkyl, amino,            haloalkyl, aminoalkyl, BOC-aminoalkyl, and cycloalkyl;    -   X₁₂ is H, hydroxyl, alkylamino, alkylamido or hydroxyalkyl;    -   X₁₃ and X₁₄ represent independently hydrogen, hydroxyl, amino,        amido, azido, halo, alkyl, alkoxy, carboxy, nitrilo, nitro,        trifluoro, aryl, alkaryl, thio, thioester, thioether, —OCOPh,        —OC(=S)OPh or both X₁₃ and X₁₄ are oxygens connected to >C=S to        form a 5-membered ring, or X₁₂ and X₁₃ form the ring of formula        (III):

where R′ and R″ represent independently an alkyl group;

R₁₂ is selected from the group consisting of hydrogen, halo, alkylether,amino, hydrazido, alkylamino, alkoxy, thioalkoxy, pyridylthio, alkenyl;alkynyl, thio, and alkylthio; and

R₁₃ is a group of the formula —NR₁₅R₁₆ wherein

R₁₅ is a hydrogen atom or a group selected from alkyl, substituted alkylor aryl-NH—C(Z)—, with Z being O, S, or NR^(a) with R^(e) having theabove meanings; wherein when R₁₅ is hydrogen than

R₁₆ is selected from the group consisting of R— and S-1-phenylethyl,benzyl, phenylethyl or anilide groups unsubstituted or substituted inone or more positions with a substituent selected from the groupconsisting of alkyl, amino, halo, haloalkyl, nitro, hydroxyl,acetoamido, alkoxy, and sulfonic acid or a salt thereof;benzodioxanemethyl, fururyl, L-propylalanyl-aminobenzyl,β-alanylamino-benzyl, T-BOC-β-alanylaminobenzyl, phenylamino, carbamoyl,phenoxy or cycloalkyl; or R₁₆ is a group of the following formula (IV):

or when R₁₅ is an alkyl or aryl-NH—C(Z)—, then, R₁₆ is selected from thegroup consisting of heteroaryl-NR^(a)—C(Z)—, heteroaryl-C(Z)—,alkaryl-NR^(a)—C(Z)—, alkaryl-C(Z)—, aryl-NR—C(Z)—and aryl-C(Z)—; Zrepresenting an oxygen, sulfor or amine.

Exemplary A₃AR agonist (disclosed in U.S. Pat. No. 5,688,774 at column4, lines 67-column 6, line 16; column 5, lines 40-45; column 6, lines21-42; column 7, lines 1-11; column 7, lines 34-36; and column 7, lines60-61):

-   N⁶-(3-iodobenzyl)-9-methyladenine;-   N⁶-(3-iodobenzyl)-9-hydroxyethyladenine;-   R—N⁶ (3-iodobenzyl)-9-(2,3-dihydroxypropyl)adenine;-   S—N⁶-(3-iodobenzyl)-9-(2,3-dihydroxypropyeadenine;-   N⁶-(3-iodobenzyladenin-9-yl)acetic acid;-   N⁶-(3-iodobenzyl)-9-(3-cyanopropyl)adenine;-   2-chloro-N⁶-(3-iodobenzyl)-9-methyladenine;-   2-amino-N⁶-(3-iodobenzyl)-9-methyladenine;-   2-hydrazido-N⁶-(3-iodobenzyl)-9-methyladenine;-   N⁶-(3-iodobenzyl)-2-methylamino-9-methyladenine;-   2-dimethylamino-N⁶-(3-iodobenzyl)-9-methyladenine;-   N⁶-(3-iodobenzyl)-9-mcthyl-2-propylaminoadenine;-   2-hexyl amino-N⁶-(3-iodobenzyl)-9-methyladenine;-   N⁶-(3-iodobenzyl)-2-methoxy-9-methyladenine;-   N⁶-(3-iodobenzyl)-9-methyl-2-methylthioadenine;-   N⁶-(3-iodobenzyl)-9-methyl-2-(4-pyridylthio)adenine;-   (1S, 2R, 3S,    4R)-4-(6-amino-2-phenylethylamino-9H-purin-9-yl)cyclopentane-1,2,3-triol;-   (1S, 2R, 3S,    4R)-4-(6-amino-2-chloro-9H-purin-9-yl)cyclopentane-1,2,3-triol;-   (±)-9-[2α,3αa-dihydroxy-4β-(N-methylcarbamoyl)cyclopent-1β-yl)]-N⁶-(3-iodobenzyl)-adenine;-   2-chloro-9-(2′-amino-2′,3′-dideoxy-β-D-5′-methyl-arabino-furonamido)-N⁶-(3-iodobenzyl)adenine;-   2-chloro-9-(2′,3′-dideoxy-2′-fluoro-β-D-5′-methyl-arabino    furonamido)-N⁶-(3-iodobenzyl)adenine;-   9-(2-acetyl-3-deoxy-β-D-5-methyl-ribofuronamido)-2-chloro-N⁶(3-iodobenzyl)adenine;-   2-chloro-9-(3-deoxy-2-methanesulfonyl-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)adenine;-   2-chloro-9-(3-deoxy-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)adenine;-   2-chloro-9-(3,5-1,1,3,3-tetraisopropyldisiloxyl-β-D-5-ribofuranosyl)-N⁶-(3-iodobenzyl)adenine;-   2-chloro-9-(2′,3′-O-thiocarbonyl-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)adenine;-   9-(2-phenoxythiocarbonyl-3-deoxy-β-D-5-methyl-ribofuronamido)-2-chloro-N⁶-(3-iodobenzyl)adenine;-   1-(6-benzylamino-9H-purin-9-yl)-1-deoxy-N,4-dimethyl-β-D-ribofuranosiduronamide;-   2-chloro-9-(2,3-dideoxy-β-D-5-methyl-ribofuronamido)-N⁶    benzyladenine;-   2-chloro-9-(2′-azido-2′,3′-dideoxy-β-D-5′-methyl-arabino-furonamido)-N⁶-benzyladenine;-   2-chloro-9-(β-D-erythrofuranoside)-N⁶-(3-iodobenzyl)adenine;-   N⁶-(benzodioxanemethyl)adenosine;-   1-(6-furfurylamino-9H-purin-9-yl)-1-deoxy-N-methyl-β-D-ribofuranosiduronamide;-   N⁶-[3-(L-prolylamino)benzyl]adenosine-5′-N-methyluronamide;-   N⁶-[3-(β-alanylamino)benzyl]adenosine-5′-N-methyluronamide;-   N⁶-[3-(N-T-Boc-β-alanylamino)benzyl]adenosine-5′-N-methyluronamide-   6-(N′-phenylhydrazinyl)purine-9-β-ribofuranoside-5′-N-methyluronamide;-   6-(O-phenylhydroxylamino)purine-9-β-ribofuranoside-5′-N-methyluronamide;-   9-(β-D-2′,3′-dideoxyerythrofuranosyl)-N⁶-[(3-β-alanylamino)benzyl]adenosine;-   9-(β-D-erythrofuranoside)-2-methylamino-N⁶-(3-iodobenzyl)adenine;-   2-chloro-N-(3-iodobenzyl)-9-(2-tetrahydrofuryl)-9H-purin-6-amine;-   2-chloro-(2′-deoxy-6′-thio-L-arabinosyl)adenine; and-   2-chloro-(6′-thio-L-arabinosyl)adenine.

Other exemplary A₃AR agonists, disclosed in U.S. Pat. No. 5,773,423, arecompounds of the formula (V):

wherein

X₁ is R^(a)R^(b)NC(=O) wherein R^(a) and R^(b) may be the same ordifferent and are selected from the group consisting of hydrogen, C₁-C₁₀alkyl, amino, C₁-C₁₀ haloalkyl, C₁-C₁₀ aminoalkyl, and C₃-C₁₀cycloalkyl;

R₂ is selected from the group consisting of hydrogen, halo, C₁-C₁₀alkyoxy, amino, C₂-C₁₀ C2alkenyl, and C₂-C₁₀ alkynyl; and

R₅ is selected from the group consisting of R— and S-1-phenylethyl, anunsubstituted benzyl group, and a benzyl group substituted in one ormore positions with a substituent selected from the group consisting ofC₁-C₁₀ alkyl, amino, halo, C₁-C₁₀ haloalkyl, nitro, hydroxy, acetamido,C₁-C₁₀ alkoxy, and sulfo.

More specific compounds include those of the above formula wherein R^(a)and R^(b) may be the same or different and are selected from the groupconsisting of hydrogen and C₁-C₁₀ alkyl, particularly when R₂ ishydrogen or halo, especially hydrogen.

Additional specific compounds are those compounds wherein R^(a) ishydrogen and R₂ is hydrogen, particularly when R₅ is unsubstitutedbenzyl.

More specific compounds are such compounds wherein R^(b) is a C₁-C₁₀alkyl or C₃-C₁₀ cycloalkyl, particularly a C₁-C₁₀ alkyl, and moreparticularly methyl.

Especially specific are those compounds where R^(a) is hydrogen, R^(b)is C₁-C₁₀ alkyl or C₃-C₁₀ cycloalkyl, and R₅ is R— or S-1-phenylethyl ora benzyl substituted in one or more positions with a substituentselected from the group consisting of halo, amino, acetamido, C₁-C₁₀haloalkyl, and sulfo, where the sulfo derivative is a salt, such as atriethylammonium salt.

An example of an especially preferred compound disclosed in U.S. Pat.No. 5,773,423 isN⁶-(3-iodobenzyl)-2-methylamino-9-[5-(methylamido)-β-D-ribofuranosyl]-adenine,also known as N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide or knownas 1-Deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purine-9-yl]-N-methyl-D-ribofuranuronamide,or by the abbreviation IB-MECA.

In addition, those compounds in which R₂ is a C₂-C₁₀ alkenylene of theformula R^(d)—C=C— where R^(d) is a C₁-C₈ alkyl are also particularlynoted in U.S. Pat. No. 5,773,423.

Also specific are those compounds wherein R₂ is other than hydrogen,particularly those wherein R₂ is halo, C₁-C₁₀ alkylamino, or C₁-C₁₀alkylthio, and, more preferably, when additionally R^(a) is hydrogen,R^(b) is a C₁-C₁₀ alkyl, and/or R₅ is a substituted benzyl.

Further exemplary A₃AR agonists disclosed in U.S. Pat. No. 5,773,423 aremodified xanthine-7-ribosides having the formula (VI):

wherein

X is O;

R₆ is R^(a)R^(b)NC(=O), wherein R^(a) and R^(b) may be the same ordifferent and are selected from the group consisting of hydrogen, C₁-C₁₀alkyl, amino, C₁-C₁₀ haloalkyl, C₁-C₁₀ aminoalkyl, and C₃-C₁₀cycloalkyl;

R₇ and R₈ may be the same or different and are selected from the groupconsisting of C₁-C₁₀ alkyl, R—and S-1-phenylethyl, an unsubstitutedbenzyl group, and a benzyl group substituted in one or more positionswith a substituent selected from the group consisting of C₁-C₁₀ alkyl,amino, halo, C₁-C₁₀ haloalkyl, nitro, hydroxy, acetamido, C₁-C₁₀ alkoxy,and sulfo; and

R₉ is selected from the group consisting of halo, benzyl, phenyl, andC₃-C₁₀ cycloalkyl.

WO 99/06053 discloses in examples 19-33 compounds selected from:

N⁶-(4-biphenyl-carbonylamino)-adenosine-5′-N-ethyluronamide:

N⁶-(2, 4-dichlorobenzyl-carbonylamino)-adenosine-5′-N-ethyluronamide;

N⁶-(4-methoxyphenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;

N⁶-(4-chlorophenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;

N⁶-(phenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;

N⁶-(benzylcarbamoylamino)-adenosine-5′-N-ethyluronamide;

N⁶-(4-sulfonamido-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;

N6-(4-acetyl-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;

N⁶-((R)-α-phenylethylcarbamoyl)-adenosine-5′-N-ethyluronamide;

N⁶-((S)-α-phenylethylcarbamoyl)-adenosine-5′-N-ethyluronamide;

N⁶-(5-methyl-isoxazol-3-yl-carbamoyl)-adenosine-5′-N-ethyluronamide;

N⁶-(1,3,4-thiadiazol-2-yl-carbamoyl)-adenosine-5′-N-ethyluronamide;

N⁶-(4-n-propoxy-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;

N⁶-bis-(4-nitrophenylcarbamoyl)-adenosine-5′-N-ethyluronamide; and

N⁶-bis-(5-chloro-pyridin-2-yl-carbamoyl)-adenosine-5′-N -ethyluronamide.

More specifically disclosed compounds include:

2-chloro-N⁶-(3-iodobenzyl)-9-[5-(methylamido)-β-D-ribofuranosyl]-adenine also known as2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide or by theabbreviation Cl-IB-MECA;

N⁶-(3-iodobenzyl)-2-methylamino-9-[5-(methylamido)-β-D-ribofuranosyl]-adenine,also known as N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide or knownas1-Deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purine-9-yl]-N-methyl-D-ribofuranuronamideor by the abbreviation IB-MECA;

N⁶-2-(4-aminophenyl)ethyladenosine (APNEA);

N⁶-(4-amino-3-iodobenzyl) adenosine-5′-(N-methyluronamide) (AB-MECA).

In one particular embodiment, Cl-IB-MECA is used in reducing ectopiclipid accumulation, accordance with the present disclosure.

When referring to “A₃AR allosteric enhancement” it is to be understoodas referring to the positive regulation, activation or incense of thereceptor activity by binding of the allosteric effector molecule at thereceptor's allosteric site which may he different from the binding siteof the endogenous ligand or agonist thereof.

In one embodiment, “enhancement” denotes an effect of the effectorcompound on the receptor exhibited by an increase of at least 15% in theefficacy of the A₃ adenosine receptor by binding of the effectorcompound to the allosteric site of the receptor and/or by a decrease indissociation rate of adenosine or an A₃AR agonist to the orthostericbinding site.

In one embodiment, the enhancement is by an “A₃AR allosteric enhancer”or “A₃ARAE” that is an imidazoquinoline derivative.

In one embodiment the A₃AR enhancer, or imidazoquinoline derivative hasthe following general formula (VII):

wherein:

-   -   R₁ represents an aryl or alkaryl being optionally substituted at        the aromatic ring with one or more substituents selected from        the group consisting of C₁-C₁₀ alkyl, halo, C₁-C₁₀ alkanol,        hydroxyl, C₁-C₁₀ acyl, C₁-C₁₀ alkoxyl; C₁-C₁₀-alkoxycarbony,        C₁-C₁₀ alkoxylalkyl; C₁-C₁₀ thioalkoxy; C₁-C₁₀ alkylether,        amino, hydrazido, C₁-C₁₀ alkylamino, pyridylthio, C₂-C₁₀        alkenyl; C₂-C₁₀ alkynyl, thio, C₁-C₁₀ alkylthio, acctoamido,        sulfonic acid; or said substituents can form together a        cycloalkyl or cycloalkenyl fused to said aryl, the cycloalkyl or        cycloalkenyl optionally comprising one or more heteroatoms;        provided that said aryl is not an unsubstituted phenyl group;    -   R₂ represents hydrogen or a substituent selected from the group        consisting of C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl; C₂-C₁₀ alkynyl,        C₄-C₁₀ cycloalkyl, C₄-C₁₀ cycloalkenyl, a five to seven membered        heterocyclic aromatic ring, C₅-C₁₅ fused cycloalkyl, bicyclic        aromatic or heteroaromatic rings; C₁-C₁₀ alkylether, amino,        hydrazido, C₁-C₁₀ alkylamino, C₁-C₁₀ alkoxy,        C₁-C₁₀-alkoxycarbony, C₁-C₁₀ alkanol, C₁-C₁₀ acyl, C₁-C₁₀        thioalkoxy, pyridylthio, thio, and C₁-C₁₀ alkylthio, acetoamido        and sulfonic acid;

and pharmaceutically acceptable salts thereof.

According to some embodiments, the R₁ substituent in the A₃ARAE has thefollowing general formula (VIII):

wherein n is 0 or an integer selected from 1-5; preferably, n is 0, 1 or2; and

-   -   X₁ and X₂ which may be the same or different, are selected from        hydrogen halogen, alkyl, alkanol or alkoxy, indanyl, pyrroline        provided that when said n is 0, X₁ and X₂ are not hydrogen.

In yet some further embodiments, R₁ in A₃ARAE is a substituent havingthe above formula (VIII), wherein X₁ or X_(2,) which may be the same ordifferent, are selected from hydrogen, chloro, methoxy, methanol or asubstituent having the formulae (VIIIa) or (VIIIb):

wherein Y is selected from N or CH.

In some yet further embodiments R₂ in A₃ARAE is selected from H, C₁₋₁₀alkyl, C₄₋₁₀ cycloalkyl, the alkyl chain may be a straight or branchedor form a four to seven membered cycloalkyl ring.

In one embodiment, R₂ in A₃ARAE is selected from a five to sevenmembered heterocyclic aromatic ring.

In some embodiments, R₂ substituents in A₃ARAE are selected from H,n-pentyl, or a five membered heterocyclic aromatic ring having thefollowing formula (IX):

wherein Z is selected from O, S or NH, preferably O.

In accordance with one embodiment R₂ in A₃ARAE comprises one or morefused rings, particularly so as to form bicyclic substituents.

Non-limiting examples of hicyclic compounds which may be used to formthe substituents in the context of the invention comprisebicyclo[2.2.1]heptane, bicyclo[4.1.0]heptane,bicyclo[4.1.0]heptan-3-carboxylic acid, bicyclo[3.1.0]hexan-3-carboxylicacid, bicyclo[4.1.0]heptan-2-carboxylic acid,bicyclo[3.1.0]hexan-2-carboxylic acid, andbicyclo[2.2.1]heptan-2-carboxylic acid.

In accordance with yet some other embodiments, R₂ in A₃ARAE may beselected from 2-cyclohexene and 3-cyclohexene.

Specific imidazoquinoline derivatives which may be used as allostericeffectors of the A₃AR are listed below:

N-(4-Methyl-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine

N-(4-Methoxy-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine

N-(3,4-Dichloro-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine

N-(4-Chloro-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine

N-(3-Methanol-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine

N-([3,4-c]Indan)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine

N-(1H-indazol-6-yl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine

N-(4-Methoxy-benzyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine

N-(1 H-Indol-6-yl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine

N-(Benzyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine

N-(Phenylethyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine

N-(3,4-Dichloro-phenyl)-2-cycloheptyl-1H-imidazo[4,5-c]quinolin-4-amine

N-(3,4-Dichloro-phenyl)-2-furyl-1H-imidazo[4,5-c]quinolin-4-amine

N-(3,4-Dichloro-phenyl)-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-4-amine

N-(3,4-Dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine

N-(3,4-Dichloro-phenyl)-2-1H-imidazo[4,5-c]quinolin-4-amine

N-(3,4-Dichloro-phenyl)-2-pentyl-1H-imidazo[4,5-c]quinolin-4-amine.

The above imidazoquinoline derivatives are regarded as allostericeffectors (modulating the activity) as they were shown to have, on theone hand, reduced affinity, if any, to the orthosteric binding sites ofthe A₁ and A_(2A), A_(2B) adenosine receptors and reduced affinity tothe orthosteric binding site of the A₃ adenosine receptor, and on theother hand, high affinity to the allosteric site of the A₃ adenosinereceptor [International Patent Application No. WO07/089507, incorporatedherein by reference].

A specifically preferred imidazoquinoline derivative in accordance withthe present disclosure isN-(3,4-Dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine(also referred to at times by the abbreviation LUF6000 or CF602), beingan allosteric enhancer.

In the context of the general formulae disclosed herein, the followingmeaning for the various terms is to be considered:

The term “alkyl” is used herein to refer to a linear or branchedhydrocarbon chain having from 1 to 10 carbon atoms and more preferably 1to 6 carbon atoms including, but not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, n-heptyl, octyl and the like.

Similarly, the terms “alkenyl” and “alkynyl” denote a linear or branchedhydrocarbon chain having, respectively, from 2 to 10, or from 3 to 10carbon atoms and more preferably 2 to 6 or 3 to 6 carbon atoms, thealkenyl or alkynyl having at least one unsaturated bond.

The alkyl, alkenyl or alkynyl substituents may be substituted with aheteroatom containing group. Thus, it should be understood that whilenot explicitly stated, any of the alkyl modifications definedhereinabove and below, such as alkylthio, alkoxy, akanol, alkylamineetc, also include the corresponding alkenyl or alkynyl modifications,such as, akenylthio, akenyloxy, alkenol, alkenylamine, or respectively,akynylthio, alkynyloxy, alkynol, alkynylamine.

The term “aryl” denotes an unsaturated aromatic carbocyclic group offrom 5 to 14 carbon atoms having a single ring (e. g., phenyl) ormultiple condensed rings (e. g., naphthyl or anthryl). Preferred arylsinclude phenyl, indanyl, benzimidazole.

The term “alkaryl” refers to -alkylene-aryl groups preferably havingfrom 1 to 10 carbon atoms in the alkylene moiety and from 6 to 14 carbonatoms in the aryl moiety. Such alkaryl groups are exemplified by benzyl,phenethyl and the like.

The term “Substituted aryl” refers to an aromatic moiety which issubstituted with from 1 to 3 substituents as defined above. A variety ofsubstituents are possible, as appreciated by those versed in the art.Nonetheless, some preferred substituents include, without being limitedthereto, halogen, (substituted) amino, nitro, cyano, alkyl, alkoxy,acyloxy or alkanol, sulphonyl, sulphynyl.

The term “Halo” or “halogen” refers to fluoro, chloro, bromo and iodo,preferably to chloro.

The term “acyl” refers to the groups H—C(O)— as well as alkyl-C(O)—.

The term “alkanol” refers to the group —COH as well as alk-OH, “alk”denoting an alkylene, alkenylene or alkynylene chain.

The term “alkoxy” is used herein to mean —O-alkyl, including, but notlimited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy and the like.

The term “alkylthio” is used herein to mean —S-alkyl, including, but notlimited to, methylthio, ethylthio, n-propylthio, isopropylthio,n-butylthio and the like.

The term “alkoxyalkyl” is used herein to mean -alkyl-O-alkyl, including,but not limited to, methoxymethyl, ethoxymethyl, n-propoxymethyl,isopropoxymethyl, n-butoxymethyl, isobutoxymethyl, t-butoxymethyl andthe like.

The term “cycloalkyl” is used herein to mean cyclic hydrocarbon radicalsincluding, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl and the like.

The term “alkoxycarbonyl” is used herein to mean —C(O)O-alkyl,including, but not limited to, methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl and the like.

The term “fused cycloalkyl” is used herein to mean any compound orsubstituent comprising at least two aliphatic rings which are connectedat a single atom (to form a spirocyclic moiety), at two mutually bondedatoms or across a sequence of atoms (bridgehead). The fused rings mayinclude any bicyclic, tricyclic as well as polycyclic moieties. Bicyclicsubstituents are preferred in accordance with some embodiments of thepresent disclosure.

The present disclosure also makes use of physiologically acceptablesalts of an A₃AR ligand, such as the above disclosed compounds. An“physiologically acceptable salts” refers to any non-toxic alkali metal,alkaline earth metal, and ammonium salt commonly used in thepharmaceutical industry, including the sodium, potassium, lithium,calcium, magnesium, barium ammonium and protamine zinc salts, which areprepared by methods known in the art. The term also includes non-toxicacid addition salts, which are generally prepared by reacting the ligandwith a suitable organic or inorganic acid. The acid addition salts arethose which retain the biological effectiveness and qualitativeproperties of the free bases and which are not toxic or otherwiseundesirable. Examples include, inter alia, acids derived from mineralacids, hydrochloric, hydrobromic, sulfuric, nitric, phosphoric,metaphosphoric and the like. Organic acids include, inter alia,tartaric, acetic, propionic, citric, malic, malonic, lactic, fumaric,benzoic, cinnamic, mandelic, glycolic, gluconic, pyruvic, succinicsalicylic and arylsulphonic, e.g. p-toluenesulphonic, acids.

The A₃AR ligand can be administered in a single dose (one timemedication) or as a continuous treatment, for over a period of days,weeks or even months.

In one embodiment, the A₃AR ligand is used for long term treatment. Inthe context of the present disclosure, long term treatment is to beunderstood to encompassing a treatment window lasting for at least days,weeks, or months, until, for example, no medically significant level oflipid is detected at the site where lipid accumulation was detectedbefore treatment began. Further in the context of the present disclosurelong term treatment can encompass chronic treatment, e.g. long termadministration without an envisaged treatment end time point. In someembodiments, the long term treatment comprises at least one week ofdaily administration of the ligand, at times, one month treatment, attimes, at least 2, 3, 4, 5, 6, or even 12 months of daily administrationof the ligand.

When referring to “treatment” by the A₃AR ligand it is to be understoodto refer to any desired pharmacological and physiological effect thatleads to medically significant improvement in the subject's conditionsas determined by parameters known to those versed in the art. Forexample, an improvement can be determined by a decrease in at least 5%in the level of triglycerides at the target site (site of depositedfat).

In one additional or alternative embodiment, improvement can bedetermined by reduction in the target site to body weight ratio (e.g.liver to body weight ratio).

In yet one other or additional embodiment, improvement can be determinedby a change in one or more parameters indicative of the functionality ofthe target organ. For example, when the target site is the liver,improvement can be determined by decrease in ALT levels.

Further, in one other embodiment, improvement can be determined by thereduction in NAS score of inflammation. NAS score is determined byvarious components referred to as the NAFLD Activity Score (NAS) andFibrosis Staging. These include, inter alia, steatosis score, lobularinflammation score, hepatocyte ballooning and fibrosis. Total NAS scorerepresents the sum of scores for steatosis, lobular inflammation, andballooning, and ranges from 0-8. NAS scores of 0-2 are considered notdiagnostic of NASH and scores of 5-8 are considered diagnostic of NASH.

In some embodiments, the treatment is of a subject that is defined assuffering from a condition associated with fat accumulation.

The A₃AR ligand can be administered on a daily basis or with a day ormore intervals between administrations. In one embodiment, A₃AR ligandis used on a daily basis, for chronic treatment.

The A₃AR ligand can be administered systemically or locally. To thisend, the A₃AR ligand is combined with pharmaceutically acceptablecarries to form a pharmaceutical composition suitable for a specificmode of administration and comprising an effective amount of the A₃ARligand.

By the term “pharmaceutically acceptable carrier” it is meant any one ofinert, non-toxic materials, which do not react with the A₃AR ligand andwhich can be added to the ligand to facilitate its delivery to subject.

In one embodiment, the carrier is one that is acceptable for preparationof a unit dosage form for oral administration.

An oral formulation may be in the form of a pill, capsule, in the formof syrup, emulsion, an aromatic powder, and other various forms. Thecarrier is selected at times based on the desired form of theformulation. The carrier may also at times have the effect of theimproving the delivery or penetration of the active ingredient to thetarget tissue, for improving the stability of the drug, for slowingclearance rates, for imparting slow release properties, for reducingundesired side effects etc. The carrier may also be a substance thatstabilizes the formulation (e.g. a preservative), for providing theformulation with an edible flavor, etc. The carriers may be any of thoseconventionally used and is limited only by chemical-physicalconsiderations, such as solubility and lack of reactivity with the A₃ARligand, and by the route of administration. The carrier may includeadditives, colorants, diluents, buffering agents, disintegrating agents,moistening agents, preservatives, flavoring agents, andpharmacologically compatible carriers. In addition, the carrier may bean adjuvant, which, by definition are substances affecting the action ofthe active ingredient in a predictable way.

Typical examples of carriers suitable for oral administration comprise(a) suspensions or emulsions in an appropriate liquid such as CremophorRH40, or methylcellulose (c.g. Methocel A4M Premium); (b) capsules (e.g.the ordinary hard- or soft-shelled gelatin type containing, for example,surfactants, lubricants, and inert fillers), tablets, lozenges (whereinthe active substance is in a flavor, such as sucrose and acacia ortragacanth or the active substance is in an inert base, such as gelatinand glycerin), and troches, each containing a predetermined amount ofthe tragacanth as solids or granules; (c) powders; (d) solution,typically when combined with a solubilizing enhancing agent; (e)liposome formulation; and others.

The A₃AR ligand is used in an amount effective to treat the fataccumulation, namely, an amount which exhibits an effect of reducinglipid depositing in tissues which do not normally (under healthycondition) harbor such fat cells, the reduction being compared betweentwo time points, at least one after the ligand consumption. The“effective amount” can be readily determined, in accordance with thepresent disclosure, by administering to a plurality of tested subjectsvarious amounts of the A₃AR ligand and then plotting the response (forexample combining several beneficial effects) as a function of theamount. At times, the amount to be used may depend on a variety offactors such as mode of administration, age, weight, body surface area,gender, health condition and genetic factors of the subject; otheradministered drugs; etc.

The effective amount of the A₃AR ligand can be defined by a unit dosageform. The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient.

When the A₃AR ligand is an A₃AR agonist, the effective amount may, forexample, be an amount of at least about 10 mg/day, e.g. at least about10 mg in a treatment regime of once daily treatment, at least about 5 mgtwice daily, at least about 3.3 mg thrice daily, etc.).

A dose of at least about 10 mg/day may be a dose of at least about 15mg/day, at least about 20 g/day, at least about 25 mg/day. In someembodiments, the dose is 25±5 mg/day.

The total amount of A₃AR ligand given a day to a patient, irrespectiveof the number of administrations is referred to herein as a “dailytreatment dose”.

Thus, in one embodiment, A₃AR ligand is formulated in a unit dosage formfor administering of the daily treatment dose of at least 10 mg/day.Where the dosage form is intended for administering to a patient in atreatment regimen comprising n doses per day, then a unit dosage formmay comprise 1/n portion of the daily treatment dose (e.g., where theintended daily treatment dose is 20 mg and the treatment regimen istwice daily then each unit dosage form will have a dose of 10 mg; orwhere the intended daily treatment dose is 25 mg and the treatmentregimen is twice daily then each unit dosage form will have a dose of12.5 mg).

As used herein, the forms “a”, “an” and “the” include singular as wellas plural references unless the context clearly dictates otherwise. Forexample, the term “an A₃AR ligand” includes one or more compounds whichare capable of specifically affecting, directly or indirectly, fully orpartially, the activity of the A₃AR.

Further, as used herein, the term “comprising” is intended to mean thatthe composition include the recited active agent, i.e. A₃AR ligand, butnot excluding other elements, such as physiologically acceptablecarriers and excipients as well as other active agents. The term“consisting essentially of” is used to define compositions which includethe recited elements but exclude other elements that may have anessential significance on treatment of fat accumulation. “Consisting of”shall thus mean excluding more than trace elements of other elements.Embodiments defined by each of these transition terms are within thescope of this invention.

Further, all numerical values, e.g. when referring the amounts or rangesof the elements constituting the composition comprising the A₃AR ligandas an active ingredient, are approximations which are varied (+) or (−)by up to 20%, at times by up to 10% of from the stated values. It is tobe understood, even if not always explicitly stated that all numericaldesignations are preceded by the term “about”.

The invention will now be exemplified in the following description ofexperiments that were carried out in accordance with the invention. Itis to be understood that these examples are intended to be in the natureof illustration rather than of limitation. Obviously, many modificationsand variations of these examples are possible in light of the aboveteaching. It is therefore, to be understood that within the scope of theappended claims, the invention may be practiced otherwise, in a myriadof possible ways, than as specifically described hereinbelow.

NON-LIMITING EXAMPLES Example 1—Effect of CF102 on Level of ALT and TG

Male C57BL/6 mice were used as a murine experimental model.

The A₃AR agonist,2-Chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA,referred to herein by the abbreviated name CF102), was synthesized forCan-Fite BioPharma by Albany Molecular Research Inc, Albany. N.Y., USA.CF102 was used in liquid form, dissolved in dimethyl sulfoxide (DMSO),used as the vehicle. Dissolved CF102 and the DMSO vehicle wereadministered at the same volume.

Male C57BL/6 mice were injected with a single subcutaneous injection of200 μg streptozotocin (STZ) two days after birth and feeding with highfat diet after 4 weeks of age. At 6 weeks of age, the mice wererandomized into vehicle and CF102 200 mg/kg treatment groups, givenorally thrice daily. Treatment was given between 6 weeks of age and 9weeks of age.

After Study termination, liver weight was measured and Liver-to-Bodyweight ratio was calculated.

Plasma ALT and liver triglyceride were measured and histologicalanalyses were made on the liver sections for the following: Hematoxylin& Eosin staining for estimation of NAFLD Activity score and Sirius redstaining for estimation of fibrosis area.

Results:

CF102 200 μg/kg reduced liver-to-body weight ratio in NASH livers(p=0.05) (FIG. 1). Furthermore, CF102 decreased ALT levels (FIG. 2A) andTriglycerides levels in the liver (FIG. 2B).

Liver sections from the Vehicle group exhibited severe micro- andmacrovesicular fat deposition, hepatocellular ballooning andinflammatory cell infiltration. The CF102 treatment group showed asignificant decrease in steatosis, ballooning and lobular inflammationcompared to the Vehicle (FIGS. 3C and 3D, FIG. 4, respectively).

Example 2—Effect of CF102 on NAS score

Male mice were injected with 200 μg/animal STZ two days after birth.From 4 weeks of age, the mice were fed with high fat diet. At 6 weeks ofage, the mice were randomized into vehicle and CF102 200 μg/kg treatmentgroups. Treatment was given orally, thrice daily. Termination wasperformed after 9 weeks.

Hematoxylin & Eosin staining was used for estimation of NAFLD Activity.

Results:

CF102 (200 μg/kg) reduced the inflammation NAS (NAFLD Activity Score)score compared to the vehicle (FIG. 5).

1.-46. (canceled)
 47. A method for treating a subject having a fibroticliver tissue, the method comprises administering to said subject anamount of CI-IB-MECA effective in reducing fibrosis area in said livertissue, to a level indicative of a non-fibrotic tissue as evidenced bySirius red staining.
 48. The method of claim 47, wherein said fibroticliver tissue comprises progressive fibrosis.
 49. The method of claim 47,administering said CI-IB-MECA to a subject having a NAFLD Activity Score(NAS) indicative of a nonalcoholic steatohepatitis (NASH) liver alongwith said fibrotic liver tissue.
 50. The method of claim 47, whereinsaid administration comprises oral administration of said CI-IB-MECA.51. The method of claim 47, wherein said administration comprises adaily treatment with an amount of at least about 10 mg/day.
 52. Themethod of claim 51, wherein said administration is once a day.
 53. Themethod of claim 51, wherein said administration is with an amount of 5mg, twice daily.
 54. The method of claim 47, comprises chronic treatmentof said subject.
 55. The method of claim 47, wherein the amount ofCI-IB-MECA is 25±5 mg/day.